title: Purinergic P2X7 receptors mediate cell death in mouse cerebellar astrocytes in culture.
abstract: The brain distribution and functional role of glial P2X7 receptors are broader and more complex than initially anticipated. We characterized P2X7 receptors from cerebellar astrocytes at the molecular, immunocytochemical, biophysical, and cell physiologic levels. Mouse cerebellar astrocytes in culture express mRNA coding for P2X7 receptors, which is translated into P2X7 receptor protein as proven by Western blot analysis and immunocytochemistry. Fura-2 imaging showed cytosolic calcium responses to ATP and the synthetic analog 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) exhibited two components, namely an initial transient and metabotropic component followed by a sustained one that depended on extracellular calcium. This latter component, which was absent in astrocytes from P2X7 receptor knockout mice (P2X7 KO), was modulated by extracellular Mg(2+), and was sensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP also elicited inwardly directed nondesensitizing whole-cell ionic currents that were reduced by extracellular Mg(2+) and P2X7 antagonists (BBG and calmidazolium). In contrast to that previously reported in rat cerebellar astrocytes, sustained BzATP application induced a gradual increase in membrane permeability to large cations, such as N-methyl-d-glucamine and 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]diiodide, which ultimately led to the death of mouse astrocytes. Cerebellar astrocyte cell death was prevented by BBG but not by calmidazolium, removal of extracellular calcium, or treatment with the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotic-type mechanism of cell death. Since this cellular response was not observed in astrocytes from P2X7 KO mice, this study suggests that stimulation of P2X7 receptor may convey a cell death signal to cerebellar astrocytes in a species-specific manner.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Costa Rica, San José, Costa Rica (E.S.); Departments of Biochemistry (L.M.G.C., M.T.M.-P., E.G.D.) and Toxicology and Pharmacology (L.A.O., D.B., A.R.A.), Faculty of Veterinary Medicine, Complutense University of Madrid, Madrid, Spain; and Neurochemistry Research Institute, Complutense University of Madrid, Madrid, Spain (L.A.O., D.B., A.R.A., M.T.M.P., E.G.D.).
authors.forename: Elvira
authors.initials: E
authors.lastname: Salas
authors.forename: Luz María G
authors.initials: LM
authors.lastname: Carrasquero
authors.forename: Luis A
authors.initials: LA
authors.lastname: Olivos-Oré
authors.forename: Diego
authors.initials: D
authors.lastname: Bustillo
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Artalejo
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Miras-Portugal
authors.forename: Esmerilda G
authors.initials: EG
authors.lastname: Delicado
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000648:ultrastructure; D001583:Benzoxazoles / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009711:Nucleotides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D061251:Primary Cell Culture; D011806:Quinolinium Compounds / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000187:drug effects / Q000502:physiology*
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D009711:Nucleotides; D011806:Quinolinium Compounds; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; D002118:Calcium